Abstract. Waterpipe, an instrument for smoking tobacco, is of increasing use in the US. Many water- pipe smokers believe that water filters out ?toxins? and, therefore, is a safer smoking alternative to cigarettes. There is a paucity of data on the health effects of waterpipe smoking, and no federal regu- lations as to its use. Based on the knowledge that the respiratory tract is the organ most directly ex- posed to waterpipe smoke, and thus likely vulnerable to toxicity from waterpipe smoking, and that the biology of the respiratory tract epithelium is highly sensitive to the environment, our goal is to devel- op an in vivo respiratory tract epithelium-based assay sensitive to the toxicity of inhaled wa- terpipe tobacco smoke and predictive of lung health. We are purposefully focused on young adult waterpipe-only smokers. Our strategy is based on a preliminary study assessing the transcriptome of the small airway epithelium (SAE) and lung clinical parameters in n=21 young, light-use waterpipe- only smokers compared to n=19 gender and ethnicity matched healthy never smokers. Strikingly, wa- terpipe smokers had higher cough and sputum scores, lower lung diffusing capacity, and a markedly abnormal SAE transcriptome compared to the healthy nonsmokers. The questions to be addressed are: (1) can we develop a sensitive, easily carried out in vivo respiratory tract epithelial transcriptome- based assay that will serve as surrogate for abnormalities in the SAE; and (2) are the changes in the respiratory tract transcriptome assessed by this assay simply evidence of exposure or do they repre- sent respiratory toxicity that correlates with clinical parameters of lung health? To answer these ques- tions, we propose a cross-sectional study design to assess a cohort of age, gender and ethnicity matched subjects, including: waterpipe-only smokers (n=200) and never smokers (n=100). The cohort will be assessed by: (1) questionnaires to document tobacco use, other exposures, lung health, and cough and sputum scores; (2) urine cotinine and blood carboxyhemoglobin levels; (3) full lung func- tion tests; (4) chest imaging; and (5) the respiratory tract epithelial transcriptome from 2 sites, the SAE (the gold standard, but impractical for large studies) and the nasal epithelium (an epithelium exposed to inhaled smoke, to serve as a surrogate for the SAE). Using standard statistical approaches, the da- ta will be analyzed to determine: (1) abnormalities in the SAE transcriptome (aim 1); (2) abnormalities in the nasal epithelial transcriptome (aim 2); and (3) if the biologic abnormalities correlate with ab- normalities in the lung function, and if so, if the nasal epithelial transcriptome can be a surrogate for the SAE transcriptome in predicting lung toxicity (aim 3). If successful, this data will help inform the FDA of the risk to respiratory health of waterpipe smoking and provide an in vivo biomarker that can be used in future studies to assess wateripipe smoking-associated risk to lung health.